1-azabicyclo [2,2,2] octan-3-one derivatives

ABSTRACT

The present invention provides novel compounds, corresponding to formulae I and II, respectively, which are able to reactivate the apoptosis-inducing function of mutant p53 proteins. This reactivation is provided by restoration of sequence-specific DNA-binding activity and transcriptional transactivation function to mutant p53 proteins, and modulation of the conformation-dependent epitopes of the p53 protein. Accordingly, the substances according to the invention will be used in pharmaceutical compositions and methods for treatment of patients suffering from various types of tumors.

This application is a division of application Ser. No. 10/381,011, filedon Mar. 20, 2003 now U.S. Pat. No. 6,921,765, which a National Phase ofPCT/SE01/02008 filed Sep. 19, 2001, which claims priority of ProvisionalApplication No. 60/234,164 filed Sep. 20, 2000, the entire contents ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to new low molecular weight compounds,which are able to restore the apoptosis-inducing function of mutant p53.The present invention also relates to pharmaceutical compositionscomprising the new compounds, as well as methods for treating cancercomprising administrating said new compounds to a mammal in needthereof.

BACKGROUND

The most common target for mutations in tumors is the p53 gene. The factthat around half of all human tumors carry mutations in this gene issolid testimony as to its critical role as tumor suppressor. p53 haltsthe cell cycle and/or triggers apoptosis in response to various stressstimuli, including DNA damage, hypoxia, and oncogene activation (Ko andPrives, 1996; Sherr, 1998). Upon activation, p53 initiates thep53-dependent biological responses through transcriptionaltransactivation of specific target genes carrying p53 DNA bindingmotifs. In addition, the multifaceted p53 protein may promote apoptosisthrough repression of certain genes lacking p53 binding sites, andtranscription-independent mechanisms as well (Bennett et al., 1998;Gottlieb and Oren, 1998; Ko and Prives, 1996). Analyses of a largenumber of mutant p53 genes in human tumors have revealed a strongselection for mutations that inactivate the specific DNA bindingfunction of p53; most mutations in tumors are point mutations clusteredin the core domain of p53 (residues 94-292) that harbors the specificDNA binding activity (Béroud and Soussi, 1998).

Both p53-induced cell cycle arrest and apoptosis could be involved inp53-mediated tumor supression. While p53-induced cell cycle arrest couldconceivably be reversed in different ways, p53-induced cell death wouldhave advantage of being irreversible. There is indeed evidence fromanimal in vivo models (Symonds et al., 1994) and human tumors (Bardeesyet al., 1995) indicating that p53-dependent apoptosis plays a major rolein the elimination of emerging tumors, particularly in response tooncogenic signalling. Moreover, the ability of p53 to induce apoptosisoften determines the efficacy of cancer therapy (Lowe et al., 1994).Taking into account the fact that more than 50% of human tumors carryp53 mutations, it appears highly desirable to restore the function ofwild type p53-mediated growth suppression to tumors. The advantage ofthis approach is that it will allow selective elimination of tumor cellscarrying mutant p53. Tumor cells are particularly sensitive to p53reactivation, supposedly for two main reasons. First, tumor cells aresensitized to apoptosis due to oncogene activation (reviewed in (Evanand Littlewood, 1998)). Second, mutant p53 proteins tend to accumulateat high levels in tumor cells. Therefore, restoration of the wild typefunction to the abundant and presumably “activated” mutant p53 shouldtrigger a massive apoptotic response in already sensitized tumor cells,whereas normal cells that express low or undetectable levels of p53should not be affected. The feasibility of p53 reactivation as ananticancer strategy is supported by the fact that a wide range of mutantp53 proteins are susceptible to reactivation. A therapeutic strategybased on rescuing p53-induced apoptosis should therefore be bothpowerful and widely applicable.

Taken together, these findings strongly suggest that pharmacologicalrestoration of p53 function would result in elimination of tumor cells.Consequently, there is a need within this field to identify substancesand methods that will enable such restoration of p53 function.

For the above defined purpose, it has been shown that p53 is a specificDNA binding protein, which acts as a transcriptional activator of genesthat control cell growth and death. Thus, the ability of the p53 proteinto induce apoptosis is dependent on its specific DNA binding function.Mutant p53 proteins carrying amino acid substitutions in the core domainof p53, which abolish the specific DNA binding, are unable to induceapoptosis in cells. Therefore, in order to obtain such substances andmethods as defined above, reactivation of p53 specific DNA binding isessential in order to trigger p53-dependent apotosis in tumors duringpathological conditions.

SUMMARY OF THE INVENTION

The present invention provides novel compounds, corresponding toformulae I and II, respectively, which are able to reactivate theapoptosis-inducing function of mutant p53 proteins. This reactivation isprovided by restoration of sequence-specific DNA-binding activity andtranscriptional transactivation function to mutant p53 proteins, andmodulation of the conformation-dependent epitopes of the p53 protein.Accordingly, the substances according to the invention will be used inpharmaceutical compositions and methods for treatment of patientssuffering from various types of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

FIG. 1A-B shows the molecular structures of compounds PRIMA-1 andMIRA-1.

FIG. 2A-C illustrates the growth suppression of tumor cells expressingmutant p53 by substances MIRA-1 and PRIMA-1 according to the invention.

FIGS. 3A, B and C illustrates how the substances PRIMA-1 and MIRA-1according to the invention induce apoptosis in human tumor cells in amutant p53-dependent manner.

FIG. 4A-C describes how the compounds MIRA-1 and PRIMA-1 according tothe invention preserve the wild type conformation of the p53 protein.

FIG. 5 describes how the substances PRIMA-1 and MIRA-1 are able topreserve the sequence specific DNA binding of the wild type p53 proteinupon heat inactivation.

FIG. 6A-B illustrates that the substances PRIMA-1 and MIRA-1 restorewild-type conformation to mutant p53 protein in cells.

FIG. 7A-B illustrates how the substances MIRA-1 and PRIMA-1 according tothe invention reactivate mutant p53 protein for specific DNA binding.

FIG. 8 illustrates the correlation between the ability of compoundsPRIMA-1 and MIRA-1 to restore the specific DNA binding andapoptosis-inducing function of mutant p53.

FIG. 9A-C shows how PRIMA-1 and MIRA-1 restore transcriptiontransactivation function to mutant p53 in cells.

FIG. 10A-C shows how PRIMA-1 and MIRA-1 transactivate expression of p53target genes in a mutant p53 dependent manner.

FIG. 11 illustrates anti-tumor activity of PRIMA-1 in vivo.

FIG. 12A-B illustrates the growth suppression of tumor cells expressingmutant p53 by structural analogs of substances MIRA-1 and PRIMA-1.

FIG. 13A-G, analogs can restore the growth suppression function ofdifferent p53 mutants.

FIG. 14 induction of p21, MDM-2 in H1299 cells by MIRA-2, PRIMA-2 andinduction of MDM-2 in SW480 cells by PRIMA-3.

FIG. 15, growth suppression by analogs is dependent on transcriptionaltransactivation function of p53.

FIG. 16 shows the molecular structures of analogs of substances MIRA-1and PRIMA-1.

Table I illustrates how the compounds PRIMA-1 and MIRA-1 suppress thegrowth of different human tumor cell lines in a mutant p53-dependentmanner

Table II describes how the compounds PRIMA-1 and MIRA-1 inhibit colonyformation in a mutant p53-dependent manner

Table III describes how the compounds PRIMA-1 and MIRA-1 restore thetranscriptional transactivation function of mutant p53

Table IV illustrates how the compounds PRIMA-1 and MIRA-1 restore thesequence-specific DNA binding of different mutant p53 proteins

Table V describes how MIRA-3 and PRIMA-2 inhibit colony formation in amutant p53-dependent manner

Definitions

In the present invention, the following terms are used:

As disclosed herein, the terms “substance S” or “compound S” bothrelates to compounds according to formula I below:

wherein

R₂ or R₃ are the same or different and are chosen from the group ofhydrogen, C1-15 alkyl, C1-15 alkenyl, C1-15 alkynyl, C1-15 alkoxy, C1-15alkylamino, aryl, C6-15 aryl alkyl, C1-15 alkyl carboxy, C2-15 alkylalkyl carboxylate, C1-15 alkylthio, C1-15 alkyl hydroxy, wherein saidgroups optionally can be substituted with halogen. A preferred compoundis 2,2-bis(hydroxymethyl)-1-azabicyclo[2,2,2]octan-3-one, which is shownbelow:

This compound is also referred to as PRIMA-1.

Another preferred compound according to formula I is2-[(6-chloro-9H-purin-9-yl)methyl-1-azabicyclo[2.2.2]octan-3-one, whichis shown below

As disclosed herein, the terms “substance F” or “compound F” both relateto compounds according to formula II below:

wherein

R₁ is chosen from the group of hydrogen, C1-15 alkyl, C1-15 alkenyl,C1-15 alkynyl, C1-15 alkoxy, C1-15 alkylamino, aryl, C6-15 aryl alkyl,C1-15 alkyl carboxy, C2-15 alkyl alkyl carboxylate, C1-15 alkylthio,C1-15 alkyl hydroxy, wherein said groups optionally can be substitutedwith halogen. A preferred compound is1-N-Cl-Propionyloxymethyl)-maleimide, which is shown below:

This compound is also referred to as MIRA-1. Other preferred compoundsaccording to formula II are N-acetyloxy-methyl-maleimide andN-hydroxymethyl-maleimide. The term “halogen”, “halo” or “halide” refersto a chlorine, bromine or iodine atom. The term “stereoisomer” refers toa compound made up of the same atoms bonded by the same bonds but havingdifferent three-dimensional structures which are not interchangeable. Acompound of the invention may be in free form, e.g., amphoteric form, orin salt, e.g., acid addition or anionic salt, form. A compound in freeform may be converted into a salt form in an art-known manner andvice-versa.

The pharmaceutically acceptable salts of the compounds of formula I (inthe form of water, or oil-soluble or dispersible products) include theconventional non-toxic salts or the quaternary ammonium salts of thesecompounds, which are formed, e.g., from inorganic or organic acids orbases. Examples of such acid addition salts include acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, paemoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.Base salts include ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth. Also, the basic nitrogen-containinggroups may be quaternized with such agents as lower alkyl halides, suchas methyl, ethyl, propyl, and butyl chloride, bromides and iodides;dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates,long chain halides such as decyl, lauryl, myristyl and stearylchlorides, bromides and iodides, aralkyl halides like benzyl andphenethyl bromides and others.

In general, as used herein, the term “alkyl” includes the straight,branched-chain and cyclized manifestations thereof unless otherwiseindicated, particularly such moieties as methyl, ethyl, isopropyl,n-butyl, t-butyl, —CH₂-t-butyl, cyclopropyl, n-propyl, pentyl,cyclopentyl, n-hexyl, cyclohexyl and cyclohexylmethyl. The same appliesto the terms “alkenyl” and “alkynyl”. The term “aralkyl”, when used,includes those aryl moieties attached to an alkylene bridging moiety,preferably methylene or ethylene.

“Aryl” includes both carbocyclic and heterocyclic moieties of whichphenyl, pyridyl, pyrimidinyl, pyrazinyl, indolyl, indazolyl, furyl andthienyl are of primary interest; these moieties being inclusive of theirposition isomers such as, for example, 2-, 3-, or 4-pyridyl, 2- or3-furyl and thienyl, 1-, 2-, or 3-indolyl or the 1- and 3-indazolyl, aswell as the dihydro and tetrahydro analogs of the furyl and thienylmoieties. Also included within the term “aryl” are such fusedcarbocyclic moieties as pentalenyl, indenyl, naphthalenyl, azulenyl,heptalenyl, acenaphthylenyl, fluorenyl, phenalenyl, phenanthrenyl,anthracenyl, acephenanthrylenyl, aceanthrylenyl, triphenylenyl, pyrenyl,chrysenyl and naphthacenyl. Also included within the term “aryl” aresuch other heterocyclic radicals as 2- or 3-benzo[b]thienyl, 2- or3-naphtho[2,3-b]thienyl, 2- or 3-thianthrenyl, 2H-pyran-3-(or 4- or5-)yl, 1-isobenzo-furanyl, 2H-chromenyl-3-yl, 2- or 3-phenoxathiinyl, 2-or 3-pyrrolyl, 4- or 3-pyrazolyl, 2-pyrazinyl, 2-pyrimidinyl,3-pyridazinyl, 2-indolizinyl, 1-isoindolyl, 4H-quinolizin-2-yl,3-isoquinolyl, 2-quinolyl, 1-phthalazinyl, 1,8-naphthyridinyl,2-quinoxalinyl, 2-quinazolinyl, 3-cinnolinyl, 2-pteridinyl,4aH-carbazol-2-yl, 2-carbazolyl, .beta.-carbolin-3-yl,3-phenanthridinyl, 2-acridinyl, 2-perimidinyl, 1-phenazinyl,3-isothiazolyl, 2-phenothiazinyl, 3-isoxazolyl, 2-phenoxazinyl,3-isochromanyl, 7-chromanyl, 2-pyrrolin-3-yl, 2-imidazolidinyl,2-imidazolin-4-yl, 2-pyrazolidinyl, 3-pyrazolin-3-yl, 2-piperidyl,2-piperazinyl, 1-indolinyl, 1-isoindolinyl, 3-morpholinyl,benzo[b]isoquinolinyl and benzo[b]furanyl, including the positionisomers thereof except that the heterocyclic moieties cannot be attacheddirectly through their nitrogen one, two or three substituentsindependently selected from C₁₋₆ alkyl, haloalkyl, alkoxy, thioalkoxy,aminoalkylamino, dialkylamino, hydroxy, halo, mercapto, nitro,carboxaldehyde, carboxy, carboalkoxy and carboxamide.

All (C1-15) moieties are preferably (C1-6) moieties and all (C1-6)moieties such as C1-6 alkyl, C1-6 allenyl, C1-6 alkoxy, and hydroxy C1-6alkyl, are more preferably C1-3 moieties (containing 1-3 carbon atomsinstead of 1-6 carbon atoms).

A “derivative” is a substance modified by varying the chemical structureof the original substances F and/or S. Such derivatives of thesubstances may involve insertion, deletion or substitution of one ormore functional groups without fundamentally altering the essentialactivity of the substance.

A “functional mimetic” means a substance which may not contain afragment or active portion of F and S but which has some or all of theproperties of the substances F and S, in particular the property ofreactivating the apoptosis-inducing function of mutant p53 by bindingthereto. More specifically, a preferred functional mimetic may be acompound in which part or all of the structure is replaced by anotherstructure. Such a functional mimetic according to the invention willprovide a spatial arrangement of reactive chemical moieties that closelyresembles the three dimensional arrangement of active groups in thesubstances F and/or S according to the present invention. As a result ofthis similar active-site geometry, the functional mimetic has effects onbiological systems which are similar to the biological activity of theoriginal molecules.

A “functional moiety” means a non-substance F and/or S-derived molecule,for example a label, a drug, or a carrier molecule.

The term “label” as used herein means a moiety, which has been joined,either covalently or non-covalently, to the present substance in orderto provide a detectable signal. Thus, such a “label” may be detected byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include 32-P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in a ELISA),biotin, dioxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available (e.g. the substance of formula canbe made detectable, e.g. by incorporating a radiolabel into a substanceor used to detect antibodies specifically raised against the substance).

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, in a first aspect, the present invention relates tosubstances F and S capable of restoration of the wild type conformationand the sequence-specific DNA binding, transcriptional transactivation,and apoptosis-inducing functions of mutant p53 The substance S comprises2,2-bis(hydroxymethyl)-1-azabicyclo[2,2,2]octan-3-one (PRIMA-1) (FIG.1B) or a functional mimetic of said structure. The substance F comprises1-(Propoxymethyl)-maleimide (MIRA-1) (FIG. 1A) or a functional mimeticof said structure. Thus, it is to be understood that said substancesneed not be identical to the structures of formulae I and II, but mayinclude variations, as long as the activity thereof is preserved. Thus,said substance may also be a derivative of the structures of formulae Iand II, or an active portion thereof. In this context, an “activeportion” means a portion of the substance F or substance S whichcontains less groups than the full structure of the substance above, butwhich retains the property of rescuing the function of mutant p53. Alsoit is to be understood that in the present application, the human p53 isparticularly preferred, even though p53 molecules of other origins mayalso be contemplated.

Thus, although WO 93/24525 suggested that amino acid sequences derivedfrom human p53 protein may be useful in the treatment of disordersincluding an overexpression of p53, the present invention is the firstto specify that low molecular weight compounds F and S are capable ofexerting such an effect by reactivation of the apoptosis-inducingfunction of the mutant p53 protein.

More specifically, the substance according to the invention is capableof providing said reactivation of the apoptosis-inducing function of p53by restoration of the sequence-specific DNA binding activity to mutant(defective) p53. Thus, even though WO 95/19367 suggested that thebinding of p53 to DNA binding sites may influence the expression ofapoptotis-regulating genes, the reactivation of the apoptosis-inducingfunction of mutant p53 by substances F and S has never been identifiedprior to the present invention.

In a preferred embodiment of the invention, the substance or functionalmimetic thereof is coupled to a functional moiety, which enhances thep53 reactivating effect of said substance. As mentioned above, such amoiety may be for example a label, a drug, or a carrier molecule. In oneembodiment, the functional moiety is a carrier molecule coupled to thepresent substance. In an alternative embodiment, the functional moietyis a p53 reactivating molecule.

Thus, in one embodiment, the present substance is coupled to a label,providing a detectable signal. A wide variety of labels and conjugationtechniques are known and reported extensively in both the scientific andpatent literature. Suitable labels include various radiolabels, enzymes,substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, magnetic particles and the like.

As regards the preparation of the present substance, general methods arefound e.g. in March 1987. Once synthesized, the molecules according tothe invention can be purified according to standard procedures in theart, including HPLC purification or other chromatographic techniques,gel electrophoresis and the like, see generally Scopes, ProteinPurification, Springer-Verlag, NY, 1982. For the preparation offunctional mimetics, techniques for the development of novelcompositions of higher potency and/or greater bioavailability and/orgreater stability than the original substances can be found e.g. inMarch 1987.

WO 95/17213 relates to molecules binding to the same DNA as p53 does,whereby the transcription thereof may be activated. Thus, although itrelates to activation of transcription of p53-regulated genes, WO95/17213 solves another problem than the present invention by use ofdifferent molecules.

WO 97/14794 and a publication by Foster et al., (1999) also relates tothe problem of how to activate the sequence-specific DNA bindingactivity of latent p53. To obtain this, a fragment of the C-terminalregulatory domain of p53 or low weight compounds are used. However, theC-terminal regulatory domain (WO 97/14794) was used to activate wildtype but not mutant p53 protein, as the present invention describes.Moreover, low molecular weight synthetic compounds which have apharmacophore different from that described in Foster et al. (1999) areforming the basis of the present invention.

Accordingly, low molecular weight compounds have been identified thatcan be used to reactivate the apoptosis-inducing function of p53. Therestoration of mutant p53 function can be achieved in living cells upontreatment of the cells with the substances in tissue culture media. Inaddition, it has also been found that the substances F and S are capableof reactivating the sequence-specific DNA binding activity of p53.Substances F and S are shown to restore p53 DNA binding in vitro and thetransactivation function of p53 in living cells.

In a second aspect, the present invention relates to a pharmaceuticalcomposition comprising a substance according to the invention, and asdefined above, together with a pharmaceutically acceptable carrier. Thepharmaceutical compositions according to the invention, and for use inaccordance with the invention, may comprise, in addition to one of theabove substances, a pharmaceutically acceptable excipient, buffer orstabilizer, or any other material well known to those skilled in the artand appropriate for the intended application. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. Examples of techniques and protocols to this end may e.g. befound in Remingtonís Pharmaceutical Sciences, 16th edition, Osol, A.(ed.), 1980.

The composition according to the invention may be prepared for any routeof administration, e.g. oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, or intraperitoneal. The precise nature of thecarrier or other material will depend on the route of administration.For a parenteral administration, a parenterally acceptable aqueoussolutions is employed, which is pyrogen free and has requisite pH,isotonicity, and stability. Those skilled in the art are well able toprepare suitable solutions and numerous methods are described in theliterature (for a brief review of methods of drug delivery, see Langer,Science 249:1 527-1533 (1990)). Preservatives, stabilizers, buffers,antioxidants and/or other additives may be included, as required. Dosagelevels can be determined by those skilled in the art, taking intoaccount the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors. Examples of the techniques and protocols mentioned above can befound in Remingtonís Pharmaceutical Sciences, 16th edition, Osol, A.(ed), 1980.

In another embodiment, the composition according to invention furthercomprises one or more additional p53 reactivators.

A third object of the present invention is the use of substances definedabove as a medicament. In particular, the invention relates to the useof these substances as medicaments for treatment of human tumors ofdifferent origin containing mutant or wild type p53 protein.Consequently, the present invention also relates to the use of the abovedefined substances in the manufacture of a medicament for the treatmentof human tumors.

Another aspect of the invention is a method of screening for compoundscapable of restoring the growth suppression function to mutant p53proteins, wherein substances according to the invention are used. Thepresent substances may be used in methods screening for: compoundshaving one or more of the biological activities of the substancesdescribed above or compounds which are binding to the same site on a p53molecule. Conventionally, the candidate compounds can be selected from asynthetic combinatorial library. Examples of screening procedures formimetics include:

-   -   A. Seeding cells expressing mutant p53 under the control of a        regulatable promoter (by for example tetracycline) in 96 well        microtiter plates, followed by treatment with a library of low        molecular weight compounds, and detecting growth suppression by        candidate compounds using the WST-1 reagent (Roche) 24-48 hours        after treatment.    -   B. Using the substances and a library of unlabelled candidate        compounds to find candidate compounds that synergize with the        substances in the restoration of the growth suppression function        to mutant p53;

A further aspect of the invention is a method of designing an organiccompound capable of reactivation of the growth suppression function ofmutant p53 proteins, wherein a substance according to the invention isused as a “lead” compound. In a preferred embodiment thereof, theorganic compound is modelled to resemble the three dimensional structureof formulae I, or/and of formulae II or a shorter fragment of thesestructures, which preferably exhibits substantially the same activity.

The design of mimetics to a known pharmaceutically active compound is aknown approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable when the active compound is difficultor expensive to synthesise or has a toxic effect, or when it isunsuitable for a particular method of administration. Mimetic design,synthesis and testing are generally used to avoid random screening of alarge number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. This can be done by systematicallyvarying the side groups in the substance, e.g. by substituting eachgroup in turn. The parts of substances constituting the active region ofthe compound are known as its “pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources (spectroscopictechniques, X-ray diffraction data and NMR). Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful when a ligand and/or binding partner change conformation uponbinding, allowing a model to take account of this in the design of themimetic.

A template molecule is then selected onto which chemical groups thatmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conventionally be selected so thatthe mimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimization ormodification can be carried out to arrive at one or more final mimeticsfor in vivo or clinical testing.

In this context, methods described in the literature allow efficientscreening of libraries for desired binding activities (see Pluckthun &Ge, Angew. Chem. Int. Ed. Engl. 30, 296-298 (1991); U.S. Pat. Nos.5,733,731; 5,872,015; and 5,612,895 Further, algorithms for threedimensional data base comparisons are available in the literature, seee.g., Cooper, et al., J. Comput.-Aided Mol. Design, 3: 253-259 (1989)and references cited therein; Brent, et al., J. Comput.-Aided Mol.Design, 2: 311-310 (1988) and references cited therein. Commercialsoftware for such searches is also available from vendors such as DayLight Information Systems, Inc., Irvine, Calif. 92714, and MolecularDesign Limited, 2132 Faralton Drive, San Leandro, Calif. 94577.

Finally, the present invention also relates to methods of medicaltreatment wherein the substances according to the invention are used.

DETAILED DESCRIPTION OF THE DRAWING

FIG. 1 shows structural formulas of 1-(Propoxymethyl)-maleimide (A) and2,2-bis(hydroxymethyl)-1-azabicyclo[2,2,2]octan-3-one (B).

FIG. 2 illustrates how substances MIRA-1 and PRIMA-1 suppressed thegrowth of cells expressing mutant p53 but did not affect the growth ofcells lacking p53 expression. More specifically, FIG. 2A shows howMIRA-1 and PRIMA-1 compound suppress growth of Saos-2 His-273 cellsexpressing mutant p53. In contrast, the effect of treatment on Saos-2cells lacking p53 expression was rather minor. The graph illustrates thedifference between viability of cells treated by compounds MIRA-1 andPRIMA-1 in the presence and absence of mutant p53, expressed as thepercentage of reduction of WST-1 cell proliferation reagent incomparison with untreated cells. The degree of WST-1 reduction, whichreflects a number of living cells, was measured by microplate reader atλ490 nm according to manufacturer (Roche). The growth suppression wascalculated as a difference in absorbance at λ490 nm between untreatedand treated cells and expressed in a percent from untreated control.Growthsuppression=100%×(control_(absorbance)−treated_(absorbance))/control_(absorbance).Two compounds were identified, compound MIRA-1 and PRIMA-1, thatsuppressed the growth of cells expressing mutant p53 but did not affectthe growth of cells lacking p53 expression. FIG. 2B shows that PRIMA-1suppresses growth of 3 cell lines expressing His-273 and His-175 mutantsof p53 under control of doxycycline-dependent promoter. In these threecell lines PRIMA-1 shows growth suppression effect on cells in a mutantp53-dependent manner. FIG. 2C shows growth curves of PRIMA-1-treatedSaos-2-His-273 cells in the absence or presence of mutant p53. FIG. 2Dshows that compounds PRIMA-1 and MIRA-1 suppress predominantly thegrowth of mutant p53 expressing cells. The ability of compounds MIRA-1and PRIMA-1 to suppress the growth was tested using 16 cell lines withdifferent p53 status: cells which do not express p53 (p53 null), cellsexpressing wild type p53 and cells expressing different mutant p53proteins. The experimental set up was as described in FIG. 2A. Thedifferences in a viability were statistically significant according toan independent t-test.

FIG. 3 illustrates the p53-dependent induction of apoptosis by PRIMA-1and MIRA-1 in Saos-2-His-273 cell line. More specifically, FIG. 3A showshow caspase inhibitors suppress the cell death induction by compoundsPRIMA-1 and MIRA-1 in Saos-2-His-273 cells. Induction of apoptosis wasdetermined by FACS analysis of ethanol fixed cells stained withpropidium iodide (PI) as percentage of a sub-G1 population. Caspaseinhibitors Z-DEVD-FMK and BOC-D-FMK (Enzyme Systems Products, CA) wereadded to Saos-2-His-273 grown in the absence of doxycycline at aconcentration 5 μg/ml prior to treatment with compounds PRIMA-1 andMIRA-1 (25 μM and 100 μM, respectively). The percentage of dead cells innon-treated cultures and in controls treated with caspase inhibitorsonly was subtracted. FIG. 3B presents TUNEL staining of Saos-2-His-273cells treated with PRIMA-1 at a concentration of 25 μM for 48 h. Hoechststaining was used to stain cell nuclei. FIG. 3C shows the induction ofapoptosis by compounds PRIMA-1 and MIRA-1 in Saos-2 and Saos-2-His-273cells. The percentage of apoptotic cells was measured by FACS analysisas it was described in Figure. A. Upper panel: apoptosis was induced inSaos-2-His-273 cells expressing p53 (no doxycycline) after 48 hours oftreatment with 10 M of MIRA-1, but not in p53-null Saos-2 cells.substances PRIMA-1 (50, 75 and 125 μM) and MIRA-1 (10 μM). Lower panel,apoptosis was induced by PRIMA-1 (50, 75 and 125 M) in mutant p53expressing Saos-2-His-273 cells, whereas in the absence of p53expression in Saos-2 cells PRIMA-1 was much less efficient.

FIG. 4 shows how compounds PRIMA-1 and MIRA-1 stabilize the native (wildtype) conformation of p53 using ELISA. More specifically, FIG. 4Aillustrates how compounds PRIMA-1 and MIRA-1 preserve theconformation-dependent PAb1620 epitope upon heat inactivation of p53proteins by incubation for 30 min at 37° C. Upper panel, GST-wild typep53 protein; middle panel, GST-His-175 mutant p53 protein; lower panel,GST-Gln-248 mutant p53 protein. Protein preparations were heated eitherin the presence or absence of PRIMA-1 and MIRA-1 and analyzed in ELISA.Absorbance of the control sample incubated on ice was taken as 100%.FIG. 4B shows how compounds PRIMA-1 and MIRA-1 prevent unfolding of p53proteins measured as appearance of PAb240 epitope in p53 proteins uponheating at 37° C. Upper panel, GST-wild type p53 protein; lower panel,GST-His-175 mutant p53 protein. FIG. 4C shows that PRIMA-1 and MIRA-1 donot affect the conformation-independent epitope DO1. No changes in D0-1epitope were observed upon incubation of p53 proteins at 37° C. Upperpanel, GST-wild type p53 protein; lower panel, GST-Gln-248 mutant p53protein.

FIG. 5 illustrates the preservation of the specific DNA binding of theGST-wild type p53 protein by the substances PRIMA-1 and F. The bandshift assay performed essentially as described before (Selivanova etal., 1996). The GST-wild type p53 protein was inactivated by 30 minincubation at 37° C. in the presence or absence of substances PRIMA-1and MIRA-1 and then tested for the DNA binding. In lanes 1 and 2,PRIMA-1 and monoclonal antibody PAb421 were added. Lane 3, inactivationof DNA binding of wtp53 by heating. Lanes 4-7 and 8-11, restoration ofthe specific DNA binding by incubation with increasing concentrations ofcompounds MIRA-1 and PRIMA-1, respectively.

FIG. 6 shows the restoration of wild-type p53 epitope PAb1620 inSKOV-His-175 cells expressing His-175 p53 mutant. PAb1620 mousemonoclonal antibody was used to detect wild type conformation of p53whereas staining with anti-p53 rabbit polyclonal antibody shows overalllevel of p53. The cell nuclei were stained with Hoechst. FIG. 6A,appearance of PAb1620 epitope after treatment with PRIMA-1. FIG. 6B,restoration of PAb1620 epitope after incubation with MIRA-1.

FIG. 7 shows the restoration of the specific DNA binding of theGST-His-175 mutant p53 protein by compounds PRIMA-1 and MIRA-1. FIG. 7ALane 1-3, GST-His-175 mutant p53 is unable to bind DNA. Lanes 4-6 andlanes 7-9, restoration of the mutant p53 specific DNA binding byincubation with increasing concentrations (45 ng, 450 ng, and 18 μg) ofcompounds MIRA-1 and PRIMA-1, respectively. PAb421 antibody was added toall reaction mixtures. FIG. 7B, compounds PRIMA-1 and MIRA-1 are able torestore the sequence-specific DNA binding of the endogenous Trp-282mutant p53 in cell extracts from Burkitt lymphoma BL-60 cells, asdetected by a band shift assay. Lane 1, endogenous Trp-282 mutant p53 incell extracts from Burkitt lymphoma BL-60 cells does not bind DNA. Lanes2 and 9, monoclonal antibodies PAb421 and/or PAb1801 do not restore theDNA binding of Trp-282 mutant p53. Incubation with increasingconcentrations (90 ng, 900 ng, and 18 μg) of compound MIRA-1 (lanes 3-5and 10-12) or compound PRIMA-1 (lanes 6-8 and 13-15) restored the DNAbinding of the Trp-282 mutant p53 protein. Monoclonal antibody PAb421was added to the reaction mixtures in lanes 2-8; PAb1801 was, added tothe reaction mixtures in lanes 9-15.

FIG. 8 illustrates the correlation between the ability of compoundsPRIMA-1 and MIRA-1 to restore the specific DNA binding andapoptosis-inducing function of mutant p53. More specifically, theapoptosis-inducing function of Phe-176 mutant p53 protein in KRC/Y renalcarcinoma cells was not restored by compounds PRIMA-1 and MIRA-1, incontrast to the His-273 mutant p53 in Saos-2-His-273 cells, as measuredby FACS analysis. The percentage of apoptotic cells was detected by FACSanalysis as it was described in FIG. 3A. Apoptosis was induced inSaos-2-His-273 cells expressing p53 (no doxycycline) after 48 hours oftreatment with substances PRIMA-1 and MIRA-1, but not in KRC/Y cells orin p53-null Saos-2 cells.

FIG. 9 demonstrates restoration of transcriptional transactivationactivity to mutant p53 by PRIMA-1 and MIRA-1. FIG. 9A, PRIMA-1 andMIRA-1 induced the wild-type p53-responsive LacZ reporter in A-431 cellscarrying His-273 mutant p53. FIG. 9B, mutant p53-dependent activation ofthe wild-type p53-responsive EGFP reporter in PRIMA-1-treatedSKOV-His-175 cells. Only cells cultured in the absence of doxycycline(express mutant p53) showed EGFP expression. FIG. 9C, MIRA-1 inducedwild-type p53-responsive EGFP reporter in SKOV-His-175 cells.

FIG. 10 demonstrates induction of p53 target genes p21 and MDM-2. FIG.10A shows induction of endogenous p21 and MDM-2 in H1299-His-175 cellstreated with 25 μM of PRIMA-1 or with 10 μM of MIRA-1. The expression ofproteins was analyzed using Western blot. FIG. 10B, shows that p53target genes in H1299-His-175 cells are induced by PRIMA-1 only in thepresence of mutant p53. FIG. 10C pictures induction of p53 target genesin PRIMA-1-treated SW480 colon carcinoma cells carrying endogenousHis-273/Ser-309 mutant p53. PRIMA-1 did not induce the same p53 targetgenes in HCT-116 colon carcinoma cells carrying wild-type p53.

FIG. 11 describes an anti-tumor activity of PRIMA-1. SCID mice wereinjected with Saos-2-His-273 cells. Intravenously (20 or 100 mg/kg) orintratumor (20 mg/kg) Injection with PRIMA-1 started 3 days afterinjection of cells and continued for 3 consecutive days two times perday. Tumor volumes were measured once in three days for two months.

FIG. 12 illustrates how structural analogs of MIRA-1 (FIG. 12A) andPRIMA-1 (FIG. 12B) suppressed the growth of cells expressing mutant p53but did not affect the cells without p53 expression. Analogs of MIRA-1:MIRA-2, MIRA-3 and analogs of PRIMA-1: PRIMA-2, PRIMA-3 inhibited thegrowth of Saos-2-His-273 cells expressing mutant p53 as measured 48hours after treatment. The graph shows that the compounds PRIMA-2,PRIMA-3 and MIRA-2, MIRA-3 selectively suppressed the growth of mutantp53-containing cells at lower concentrations than original MIRA-1 andPRIMA-1 compounds. Experimental setting was as described in FIG. 2A.

FIG. 13 shows how analogs of PRIMA-1 and MIRA-1 can suppress growth ofcells expressing different p53 mutants under control oftetracycline-dependent promoter (cell do not express mutant p53 in thepresence of doxycycline). FIG. 13A, PRIMA-2 and PRIMA-3 restored thegrowth suppression function of His-175 mutant p53 in SKOV cells. FIG.13B, PRIMA-3 reactivated the hot spot p53 mutant Trp-248 in H1299 lungadenocarcinoma cells. FIG. 13C, MIRA-2 and MIRA-3 reactivated His-175mutant of p53 in SKOV cells. FIG. 13D, MIRA-2 and MIRA-3 restore thegrowth suppression function of His-273 mutant p53 in SKOV cells. FIG.13E, MIRA-3 restored the growth suppression function of His-175 in H1299cells. FIG. 13F, PRIMA-3 restored the growth suppression function ofHis-175 mutant p53 in H1299 cells. FIG. 13G, PRIMA-2 reactivated mutantp53 His-273 in SKOV cells.

FIG. 14, Induction of p53 target genes by analogs of PRIMA-1 and MIRA-1.FIG. 14A, induction of p21 and MDM-2 in H1299-His-175 cells by MIRA-2and PRIMA-2. FIG. 14B, induction of MDM-2 in SW480-His-273 coloncarcinoma cells by PRIMA-3.

FIG. 15 Growth suppression induced by analogs is dependent ontranscriptional transactivation function of p53. FIG. 15A, Treatmentwith cycloheximide rendered H1299-His-175 resistant to treatment withMIRA-3 The effect of cycloheximide was notable in cells without mutantp53 expression (dox+), however, that effect was rather minor compare tocells expressing mutant p53 (dox−). FIG. 15B shows that mutation intransactivation domain of His-175 mutant p53 protected SKOV cellsagainst treatment with PRIMA-2.

FIG. 16 shows structural formulas of compounds MIRA-2, MIRA-3 (FIG. 13A)and PRIMA-2 and PRIMA-3 (FIG. 13B).

Experimental

Below, the present invention will be described in more detail by way ofexamples that are not intended to limit the scope of the invention inany way. All references given below and elsewhere in the presentspecification are hereby included herein by reference.

Materials and Methods

Plasmids

The plasmids encoding the GST-human wild type p53 fusion protein and theGST-human mutant p53 proteins His 175 were described earlier (Selivanovaet al., 1996). The p53-EGFP plasmid contains 13 synthetic p53 consensusDNA binding sites in front of the EGFP coding sequence. Transienttransfections experiments were performed with Lipofectamine 2000according to the manufacturer's recommendations (Invitrogen™ LifeTechnologies, Groningen, The Netherlands).

Chemical Library

A library of low molecular weight compounds was obtained from NationalCancer Institute (NCI), Bethesda, USA. For more information, see website http://dtp.nci.nih.gov

Screening of the Chemical Library and Growth Suppression Assays

Saos-2-His-273 cell line stably transfected with construct allowingexpression of mutant His-273 p53 in a tetracycline-dependent manner wasused for screening (Selivanova et al., 1997). p53 expression wasinhibited by incubation of cells with doxycycline (5 μg/ml). Cells weregrown in 96-well plates at a density of 3000 cells per well with orwithout doxycycline and treated with 25 μM of the compounds from the NCIlibrary of low molecular weight (LMW) compounds. After 48 hours ofincubation the proliferative cell reagent WST-1 (Roche) was added to thecells. The degree of WST-1 reduction, which reflects cell viability, wasmeasured by microplate reader at λ490 nm according to the manufacturer(Roche).

FACS Analysis

Cells were placed on 12-well plate at a density of 30000/cm² and treatedwith compounds. After 48 h incubation cells were harvested bytrypsinization, fixed with 70% ethanol, treated with RNase A (0.25mg/ml) and stained with propidium iodide (0.02 mg/ml). Samples wereanalyzed on a Becton Dickinson FACScan. Data were analyzed by theCellQuest software, version 3.2.1.

Colony Formation Assay

Cells were treated with the compounds PRIMA-1 and MIRA-1 and seeded inplates at 500 cells per plate. Colonies were stained with Giemsa andcounted 14 days after seeding.

Luciferase Assays

Transactivation assays using p53-responsive promoter constructs linkedto the luciferase reporter gene (PG-luc) were performed by the DualLuciferase Reporter Assay System (Promega) according to themanufacturer. Saos-2-His273 cell line stably transfected with luciferasereporter plasmid PG-luc (2 mg) was treated with compounds PRIMA-1 andMIRA-1 at concentration of 50 and 10 μM, respectively. A luciferaseactivity was assayed 1; 3.5 and 15 hours post-treatment.

DNA Binding Assays

The GST-p53 proteins were prepared as described (Selivanova et al.,1997). Band shift assays were performed in binding buffer containing 100mM HEPES pH 7.5, 50 mM KCl , 1 mg/ml BSA, 0.1% Triton X-100, 2 mM MgCl2and 1 mM DTT essentially as in (Selivanova et al., 1996).

ELISA

20 ng of GST-wtp53, GST-mtp53-175 and GST-mtp53-248 were heated at 37°C. for 30 min or kept on ice. The procedure was performed with orwithout tested compounds. The ELISA analyses were done as described by(Foster et al., 1999). Briefly, after the treatment, samples werediluted with coating buffer (150 mM KCL, 25 mM HEPES) supplemented with10 mM DTT. The whole mixture was apply to ELISA plates (MaxiSorp, Nunc)and incubated at +4° C. for 35 min. The wells were washed with coatingbuffer. The wells were blocked by 5% skim milk in PBS by incubating at+4° C. for 1 h. Wells were rinsed twice with PBS followed by addition ofmouse primary antibodies (PAb 1620 or PAb 240) diluted 1:250 in coatingbuffer. Samples were incubated at +4° C. for 30 min. Wells were rinsedtwice with PBS. After that, a secondary antibody (anti-mouse, conjugatedwith horse radish peroxidase) was incubated with samples at +4° C. for30 min. Then plates were washed 5 times with PBS and a peroxidasesubstrate was added. An absorbance at λ405 nm was monitored by ELISAreader.

TUNEL staining, immunostaining, lacZ staining, preparation of cellextracts, ELISA with cell extracts and Western blotting were performedaccording to standard procedures.

In vivo Experiments

All animal studies were approved by the local animal ethical committeeand animal care was in accordance with institutional guidelines. Fortoxicity assessment, 12 SCID mice (average weight 25 g) were divided in4 groups. Three groups received daily i.v. injections of 1, 10 and 100mg/kg of PRIMA-1 in PBS for 5 days. Control animals were injected withPBS. We measured weights of the mice for 1 month after the lastinjection. For assessment of the anti-tumor activity of PRIMA-1, 12 SCIDmice were inoculated with 1×10⁶ Saos-2-His-273 cells in 90% Matrigel(Becton Dickinson, Le Pont-De-Claix, France) subcutaneously andunilaterally into the right flanks. After 3 days the mice were dividedinto 4 groups. Two groups received i.v. injections of PRIMA-1 at a doseof either 20 or 100 mg/kg, one group received intratumor injections ofPRIMA-1 at a dose of 20 mg/kg, and the last group was used as a control.Injections were performed twice daily for 3 days. Tumor volume wasmeasured during 2 months.

Results and Discussion

Growth Suppression by Compounds PRIMA-1 and MIRA-1 Depends on Mutant p53Expression

According to the present invention, the NCI library of low molecularweight compounds has been screened for compounds that can suppress thegrowth of human tumor cells in a mutant p53-dependent manner.

Saos-2-His-273 cell line stably transfected with construct allowingexpression of mutant His-273 p53 in a tetracycline-dependent manner wasused for screening (Selivanova et al., 1997). Cells were grown in96-well plates at a density of 3000 cells per well with or withoutdoxycycline. The treatment was done at a concentration of 25 μM of eachchemical from the NCI library of low molecular weight (LMW) compounds.After 48 hours of incubation the proliferative cell reagent WST-1(Roche) was added to the cells. The degree of WST-1 reduction, which isproportional to the cell viability, was measured by a microplate readerat λ490 nm according to the manufacturer (Roche). Two compounds wereidentified which were able to suppress the growth of Saos-2-His-273cells expressing p53, but did not affect the growth of Saos-2 cellswhich do not express mutant p53 (FIG. 1A).

The ability of the compounds PRIMA-1 and MIRA-1 to suppress the growthof mutant p53-expressing cells was further evaluated using a colonyformation assay. Saos-2 or Saos-2-His-273 cells were treated withdifferent dosed of the compounds MIRA-1 and PRIMA-1 and seeded inplates. The cells were Giemsa stained and scored for the appearance ofcolonies after 14 days. As shown in Table II, treatment with 5 M of thecompound MIRA-1 dramatically reduced the number of colonies formed byHis-273 expressing Saos-2 cells (15% of untreated control), but was lessefficient in inhibiting Saos-2 cells lacking p53 (48% inhibition).Treatment with the compound PRIMA-1 was inhibitory in a mutantp53-dependent manner at higher doses, around 50-100 μM.

Next we tested the ability of compounds PRIMA-1 and MIRA-1 to suppressthe growth of tumor cells in a mutant p53-dependent manner using seriesof human tumor cell lines with different p53 status (p53 null, wild typep53, mutant p53). The human cell lines were as follows. p53 null: Saos-2osteosarcoma, K562 acute myeloid leukemia, and HL60 promyelocyticleukemia. Wild type p53 expressing cells: NHF normal human fibroblasts,HeLa cervical carcinoma (carries HPV E6 protein, leading to p53degradation), U2OS osteosarcoma, and EBV-positive IARC 171lymphoblastoid cell line. Mutant p53 expressing lines: Burkitt lymphomalines BL41 (Gln-248 mutant p53); DG75 (His-283), Raji (Gln-213,His-243), Ramos (Asp-254); BJAB (Arg-193), and Saos-2-His-273,SKOV-His-175, SKOV-His-273 and H1299-His-175 expressing p53 mutantsunder the control of doxycycline-dependent promoter. In addition, mousep53 null J3D T-cell lymphoma line was used. As could be seen in Table I,compounds MIRA-1 and PRIMA-1 suppressed the growth of mutantp53-expressing cells more efficiently then p53 null and wild type p53containing cells. The data from these experiments were summarized in agraph shown in FIG. 2B. The differences in responses between the groupsof cell lines (p53 null, wild type p53 and mutant p53) werestatistically significant as verified by an independent t-test.

As shown in FIG. 2C, PRIMA-1 completely inhibited growth ofSaos-2-His-273 cells expressing mutant p53. In the absence of mutant p53expression, PRIMA-1 only caused a minor reduction in growth rate.

Restoration of the Apoptosis-inducing Function to Mutant p53 byCompounds MIRA-1 and PRIMA-1

To address the question whether growth suppression induced by compoundsMIRA-1 and PRIMA-1 occur due to the induction of apoptosis, we testedwhether caspase inhibitors can inhibit MIRA-1 and PRIMA-1 induced growthsuppression. Saos-2-His 273 cells were treated with compounds MIRA-1 andPRIMA-1 in the presence or absence of caspase inhibitors inhibitorsZ-DEVD-FMK and BOC-D-FMK (Enzyme Systems Products, CA). Induction ofcell death was determined by FACS analysis of ethanol-fixed cellsstained with propidium iodide (PI) as percentage of sub-G1 population.As it is evident from FIG. 2A, caspase inhibitors suppressed the celldeath induced by compounds PRIMA-1 and MIRA-1. Therefore we concludethat compounds MIRA-1 and PRIMA-1 can induce apoptosis. In addition,apoptotic morphology was detected in Saos-2-His-273 cells stained withHoechst dye after treatment with compound PRIMA-1. TUNEL staining ofSaos-2-His-273-cells treated with compound PRIMA-1 also confirmedapoptosis induction (data not shown). We also observed a difference inthe kinetics of apoptosis induction by compounds PRIMA-1 and F: whereasapoptosis induced by PRIMA-1 was evident after 48 hours of treatment,compound MIRA-1 induced cell death much faster, within 6-12 hours aftertreatment (data not shown). These results suggest that compounds PRIMA-1and MIRA-1 trigger different apoptotic pathways.

We examined whether apoptosis induced by compounds PRIMA-1 and MIRA-1 isp53-dependent using Saos-2-His-273 cells grown in the presence orabsence of doxycyclin. As shown in FIG. 3B, the induction of apoptosisby compounds PRIMA-1 and MIRA-1 occurred only in the presence of p53expression. Taken together, these results clearly indicate that growthsuppression by compounds MIRA-1 and PRIMA-1 is mediated by a mutant p53and is not due to the nonspecific cellular toxicity.

Modulation of the Conformation of the p53 Core Domain by CompoundsMIRA-1 and PRIMA-1

To get insight into the molecular mechanism of compounds MIRA-1- andPRIMA-1-mediated reactivation of mutant p53, we tested whether theconformation of p53 was affected by these compounds. It has been shownthat point mutations in p53 result in destabilization of the nativeconformation of the p53 core domain, resulting in the loss of wildtype-specific conformation-dependent epitope for the monoclonal antibodyPAb1620 and appearance of a new epitope recognized by the monoclonalantibody PAb240 (Cho et al., 1994). In addition, heat denaturation ofthe wild type p53 has a similar effect. Therefore we examined whethercompounds PRIMA-1 and MIRA-1 can stabilize the native (wild type)conformation of p53. Results presented in FIG. 4A demonstrate thatcompounds PRIMA-1 and MIRA-1 preserve the conformation-dependent epitopefor PAb1620 antibody of the recombinant wild type and mutant p53proteins heated for 30 min. at 37° C. For the GST-wtp53 protein thedifference between treated and untreated samples in remaining PAb1620epitope after treatment with the compound PRIMA-1 has reachedstatistical significance at p=0.05 (n=5) according to a paired t-test.Importantly, results presented in FIG. 4B demonstrate that compoundsPRIMA-1 and MIRA-1 are able to prevent unfolding of p53 proteinsmeasured as appearance of PAb240 epitope in p53 proteins upon heating at37° C. According to a paired t-test the difference in the appearance ofPAb240 epitope between control and PRIMA-1-treated samples for theGST-wtp53 and GST mutant p53-His175 proteins reached statisticalsignificance at p=0.01 and p=0.1, respectively. FIG. 4C shows thatnon-conformational epitope in the N-terminus of p53 recognized by DO-1antibody is not affected by incubation with compounds MIRA-1 andPRIMA-1. Thus, the compounds MIRA-1 and PRIMA-1 are able to preserve thenative conformation of mutant p53 proteins.

Restoration of Wild Type p53 Conformation in vitro and in Living Cells

To test whether PRIMA-1 can convert mutant p53 into wild-type p53conformation, we used the conformation-specific antibodies PAb1620 andPAb240. Treatment of recombinant GST-wild type p53 protein with PRIMA-1resulted in a 40% increase in the PAb1620+ fraction and a correspondingdecrease in the PAb240+ fraction, while the DO-1+ fraction remainedunchanged. About 40% increase in PAb1620+ fraction and ˜20% reduction inPAb240+ fraction were observed in similar experiments with MIRA-1. Wemeasured the fraction of PAb1620+ p53 in protein extracts fromPRIMA-1-treated SKOV-His-175 cells using ELISA. After treatment with 150μM of PRIMA-1, the PAb1620+ fraction reached 146±18% (the value foruntreated cells was set to 100%), whereas the DO-1 fraction was 88±9%.This demonstrates that PRIMA-1 can stabilize mutant p53 in a wild typeconformation, both in vitro and in living cells.

Furthermore, immunostaining with PAb1620 demonstrated the ability ofPRIMA-1 to convert mutant p53 to wild type conformation in living cells.As shown in FIG. 6A, treatment of SKOV-His-175 cells with PRIMA-1resulted in the appearance of PAb1620-positive p53 in cells and aconcomitant decrease in total p53 levels according to staining withpolyclonal anti-p53 antibodies. A similar effect was observed for cellstreated with MIRA-1 (FIG. 6B).

Compounds MIRA-1 and PRIMA-1 Can Restore the Sequence-specific DNABinding of Mutant p53 Proteins

Next we addressed the question whether the restoration of theapoptosis-inducing function of mutant p53 proteins by compounds MIRA-1and PRIMA-1 operates through the specific DNA binding activity of p53.Do compounds PRIMA-1 and MIRA-1 restore the specific DNA binding of p53?We investigated the DNA binding of p53 proteins in the presence orabsence of compounds MIRA-1 and PRIMA-1 in a band shift assay, asdescribed before (Selivanova et al., 1996; Selivanova et al., 1997).Results presented in FIG. 5A demonstrate that compounds MIRA-1 andPRIMA-1 are able to restore the specific DNA binding of the GST-wildtype p53 protein inactivated by incubation at 37° C. for 30 min.Moreover, the compounds MIRA-1 and PRIMA-1 were able to restore thespecific DNA binding of the GST-His-175 mutant p53 protein, as shown inFIG. 5B. Substitution of arginin at position 175 causes a grossunfolding of the DNA binding core domain of p53. Therefore, therestoration of the DNA binding of this mutant was regarded as anexceptionally difficult task. Restoration of the DNA binding of His-175p53 mutant demonstrates a high potency of the identified compounds.Since His-175 mutant was shown to gain an oncogenic function, thisresult appears to be of particular importance. Compounds PRIMA-1 andMIRA-1 were also able to restore the sequence-specific DNA binding ofthe endogenous Trp-282 mutant p53 in cell extracts from Burkitt lymphomaBL-60 cells, as shown in FIG. 6B.

We tested the ability of compounds PRIMA-1 and MIRA-1 to restore thespecific DNA binding properties of a broad series of hot spot p53mutants, using cellular extracts of human tumor cell lines carryingdifferent p53 mutants as a source for endogenous p53 protein. Thecompound PRIMA-1 restored the specific DNA binding of 13 out of 14mutant p53 proteins tested in band shift assays, irrespective on theresidual DNA binding (see Table III). The compound MIRA-1 restored theDNA binding of 3 out of 14 mutant p53 proteins (Table III). Thus, thecompounds MIRA-1 and PRIMA-1 were not only capable of restoring the DNAbinding of recombinant mutant p53 proteins, but reactivated the DNAbinding of a number of endogenous mutant p53 proteins in cell extracts.The only exception for compound PRIMA-1 was the Phe-176 mutant, whichwas not reactivated by either of the compounds.

Taking into consideration our results that compounds MIRA-1 and PRIMA-1are not capable of restoring the specific DNA binding of the Phe-176mutant p53 protein in KRC/Y cells, we tested whether theapoptosis-inducing function of this mutant could be reactivated bycompounds MIRA-1 and PRIMA-1. KRC/Y cells were treated with 50 μM and 75μM concentrations of compounds MIRA-1 and PRIMA-1, respectively, and thepercentage of dead cells was measured by FACS analysis as describedabove. As demonstrated in FIG. 5, the induction of apoptosis in KRC/Ycells was much less prominent as compared to Saos-2-His-273 cells. Infact, the response of KRC/Y cells to treatment was comparable with thatof Saos-2 cells that do not express p53. Thus, it appears that thedefect caused by substitution of the Cys residue at position 176 isirreversible. The substitution of this Cys residue abolishes the bindingof a Zn atom which holds together the DNA-binding loops of the p53 coredomain. Therefore, the unfolding of this mutant p53 protein is probablytoo extensive to be restored.

PRIMA-1-Induced Apoptosis Depends on the Transactivation Function of p53

To further ascertain that PRIMA-1 exerts its effect through p53-mediatedtranscriptional transactivation and de novo protein synthesis, we testedthe effect of cycloheximide on PRIMA-1-induced growthinhibition/apoptosis. Pretreatment of SKOV-His-175 cells withcycloheximide before addition of PRIMA-1 caused a 4-fold increase incell survival according to the WST-1 proliferation assay. Thecycloheximide treatment renders SKOV-His-175 resistant to MIRA-1 aswell, resulting in about 4 fold increase in cell survival. Moreover, wehave found that the viability of SKOV cells carrying His-175-22/23mutant p53 that has an inactivated transactivation domain was at leasttwice as high as that of SKOV-His-175 cells after PRIMA-1 treatment. Inaddition, SKOV-His-175 cells were at least 3 fold more sensitive totreatment with MIRA-1 in comparison with SKOV-His-175-22/23 cells. Takentogether, these results provide a convincing evidence thattranscriptional transactivation by p53 is critical for PRIMA-1- andMIRA-1-induced cell death.

Compounds MIRA-1 and PRIMA-1 can Restore the TranscriptionalTransactivation Function of Mutant p53 in Living Cells

Having established that compounds MIRA-1 and PRIMA-1 can reactivate thespecific DNA binding of mutant p53 in vitro, we addressed the questionwhether compounds MIRA-1 and PRIMA-1 can restore the transcriptionaltransactivation function of mutant p53 function in living cells.Saos-2-His-273 cells carrying a p53-responsive PG-luciferase reportergene were treated with compounds MIRA-1 and PRIMA-1 and luciferaseactivity was measured using the Dual Luciferase Reporter Assay System(Promega) according to the manufacturer. As shown in Table IV, compoundsMIRA-1 and PRIMA-1 stimulated transcription of the luciferase gene 1.5-2fold. Interestingly, the kinetics of the induction of luciferase geneexpression differed between compounds MIRA-1 and PRIMA-1. Whereascompound MIRA-1 stimulated luciferase expression 2-fold already after3.5 hours, 2-fold induction by compound PRIMA-1 was achieved only after15 hours of treatment. The kinetics of induction of luciferase geneexpression correlates with the fast and slow induction of apoptosis bycompounds MIRA-1 and PRIMA-1, respectively.

Treatment of A431 cells that carry endogenous His-273 mutant p53 and atransfected p53-responsive lacZ reporter with 50 μM of PRIMA-1 for 20hours resulted in the appearance of lacZ-positive cells whereasuntreated cells were negative (FIG. 9A). Similar results were obtainedafter treatment with 5 μM of MIRA-1 for 12 hours.

We also transiently transfected SKOV-His-175 cells with a p53-responsiveEGFP reporter. FIG. 9B shows a strong induction of EGFP expression inSKOV-His-175 cells expressing mutant p53 after treatment with PRIMA-1for 24 hours. In contrast, SKOV-His-175 cells grown in the presence ofdoxycycline (p53 off) did not express detectable levels of EGFP. Theinduction of EGFP was also observed in cells treated with 5μM of MIRA-1for 24 hours (FIG. 9C).

As a final confirmation that PRIMA-1 and MIRA-1 can rescuetranscriptional transactivation of mutant p53, we examined if PRIMA-1 orMIRA-1 were able to induce two classical p53 target genes, p21 and MDM2.Treatment of H1299-His-175 cells expressing mutant p53 with eitherPRIMA-1 or MIRA-1 resulted in a solid induction of both MDM2 and p21(FIG. 10A). Importantly, treatment with, PRIMA-1 or MIRA-1 compound ofthe same cells in the absence of mutant p53 expression did not cause anyinduction of MDM2 nor p21 (FIG. 10B). In addition, both chemicalsinduced MDM2 and p21 in SW480 colon carcinoma cells carrying endogenousHis-273 mutant p53 (FIG. 10C), but did not cause any significant changesof MDM2 and p21 protein levels in HCT116 colon carcinoma cells thatcarry wild type p53.

Stimulation of transcriptional transactivation function by compoundsMIRA-1 and PRIMA-1 correlated with the data obtained in band shiftexperiments and demonstrates that compounds MIRA-1 and PRIMA-1 can workboth in vitro and in vivo as reactivators of the specific DNA bindingand transactivation functions of p53.

Toxicity and Anti-tumor Activity of PRIMA-1 in vivo

Intravenous injections of PRIMA-1 in mice did not cause any obviouschanges in behavior or weight compared with untreated control animals.The average weight of untreated control mice was 20±0.6 g (means±SE,n=3) and the average weight of mice treated with PRIMA-1 at the highestused dose of 100 mg/kg was 20±0.2 g after one month of observation. Toassess the effect of PRIMA-1 on human tumor xenografts, we inoculatedmice with Saos-2-His-273 cells expressing mutant p53. The animalsreceived intratumor (20 mg/kg) or intravenous (20 or 100 mg/kg)injections of PRIMA-1 twice a day for three days. In the untreatedcontrol group, the average tumor volume after 59 days was 555.7±284 mm³(means±SE, n=3). At this time, mice that received intravenous injectionsof PRIMA-1 at a dose of 100 mg/kg had an average tumor volume of 11.7±8mm³, and mice that treated with 20 mg/kg PRIMA-1 i.v. had an averagetumor volume of 53±48.5 mm³ (FIG. 5). Mice that got intratumorinjections of 20 mg/kg of PRIMA-1 had an average tumor volume of 5.3±2.7mm³. The differences in tumor volume between untreated control mice andanimals treated with PRIMA-1 are all statistically significant (P=0.041for intratumor injections of 20 mg/kg, P=0.066 for intravenous injectionof 20 mg/kg, and P=0.045 for intravenous injection of 100 mg/kg,according to the paired t-test for the entire observation period). Thus,PRIMA-1 has in vivo anti-tumor activity in this animal tumor model.

Identification of Structural Analogs of Compounds MIRA-1 and PRIMA-1that are Able to Specifically Suppress Growth of Mutant p53-ContainingCells.

In order to identify the active groups of the p53-reactivating compoundsPRIMA-1 and MIRA-1 a series of structural analogs of compounds MIRA-1(23 compounds) and PRIMA-1 (16 compounds) were tested. Saos-2-His-273cells grown in the presence or absence of doxycycline and parentalSaos-2 cells were treated with the structural analogs and analyzed asdescribed above. The effect of structural analogs on cell growth wastested using different concentrations of the compounds, ranging from 2.5to 25 μM. Compounds MIRA-2, MIRA-3 and PRIMA-2, PRIMA-3 were identifiedthat were able to suppress the growth of Saos-2-His-273 cells expressingmutant p53 (tet−). At the same time the identified analogs did notaffect growth of Saos-2-His-273 cells which did not express mutant p53in the presence of doxycycline (tet+) or parental cell line Saos-2lacking p53. Although the structural analogs did not possess higheractivity than the originally identified compounds, compounds PRIMA-2 andPRIMA-3 selectively suppressed the growth of mutant p53 containing cellsat lower concentrations (2.5 μM and 5 μM, respectively), then it wasrequired to achieve for the selective effect by compound PRIMA-1 (10μM). In addition, compounds MIRA-2 and MIRA-3 suppressed the growth ofcells in a mutant p53-dependent manner at 10 μM concentration, incontrast to 25 μM of compound MIRA-1 required for the selective effect.

Structural Analogs of PRIMA-1 and MIRA-1 can Restore the GrowthSuppression Function of Three Hot Spot p53 Mutants in a Tumor Cells ofDifferent Origin.

We have examined whether selected analogs can restore the growthsuppression function of different p53 hot spot mutants using cell lineswith doxycycline-dependent expression of His-175 and Gln-248 p53mutants. Growth suppression by analogs in the presence or absence of p53expression was measured as described in FIG. 1. As demonstrated in FIG.13A, analogs of PRIMA-1, PRIMA-2 and PRIMA-3 restored the growthsuppression function to His-175 mutant p53 in SKOV-His-175 cells.Reactivation of another hot spot p53 mutant Gln-248 by PRIMA-3 compoundis shown in FIG. 13B. Analogs of MIRA-1 induced mutant p53-dependentgrowth suppression in SKOV-His-175 cells (FIG. 13C). Notably, theconcentration of the analogs required to achieve the p53-dependentsuppression of growth was lower for the analogs in all cases, indicatinghigher potency of the analogs.

Next we address the question whether the p53-dependent growthsuppression effect by analogs of PRIMA-1 and MIRA-1 could be reproducedin tumor cell lines of different origin. We compared growth inhibitionby MIRA-1 analogs in two tumor cells lines expressing His-273 p53mutant, the osteosarcoma line Saos-2-His-273 and ovarian carcinoma lineSKOV-His-273, and found restoration of the His-273 mutant in both tumorlines (FIG. 13D). The growth of His-175 mutant p53-expressingH1299-His-175 lung carcinoma cells was inhibited by MIRA-3 in a mutantp53-dependent manner as well (FIG. 13E). Thus, reactivation of mutantp53 by MIRA-1 analogs does not depend on the origin of tumor cells.

The same conclusion was reached in experiments using PRIMA-1 analogs.Both PRIMA-2 and PRIMA-3 reactivated the His-175 mutant in SKOV andH1299 cells (FIG. 13F). Restoration of His-273 mutant function in bothSaos-2 and SKOV cells by PRIMA-2 is shown in FIG. 13G.

We assessed the effect of analogs on the long term survival of tumorcells in the presence or absence of p53 expression in a colony formationassay. As shown in Table V, PRIMA-2 and MIRA-3 suppressed the formationof colonies in H1299-His-175 cells in a mutant p53-dependent manner.

Thus, analogs of MIRA-1 and PRIMA-1 are able to restore the growthsuppression function of the three most common hot spot p53 mutants atlower concentration that the original compounds and independently ofgenetic background.

Rescue of the Transcriptional Transactivation Function of Mutant p53 byAnalogs.

In order to assess the effect of the analogs on the transcriptionaltransactivation function of mutant p53, we tested the expression of p53target genes after treatment with analogs of colon carcinoma cells SW480expressing endogenous His-273 mutant p53. As demonstrated using Westernblotting, PRIMA-2 and PRIMA-3, as well as MIRA-2 and MIRA-3 inducedexpression of the p53 target genes p21 and MDM-2 in a mutantp53-dependent manner (FIG. 14). Thus, analogs of PRIMA-1 and MIRA-1 areable to restore the transcription transactivation function of mutantp53.

We next addressed the question whether growth suppression by the analogsis dependent on p53-mediated transcriptional transactivation. Treatmentof H1299-His-175 cells with cycloheximide prevented growth suppressionby MIRA-3, indicating the requirement of new protein synthesis (FIG.15A). As shown in FIG. 15B, the growth suppression effect of PRIMA-2 wascompletely abolished in H1299-His175-22/23 cells, expressing atranscriptionally defective mutant p53 carrying substitutions atpositions 22 and 23 in addition to 175. These results allow us toconclude that the transcriptional transactivation function of mutant p53is required for the suppression effect of the analogs.

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1. A pharmaceutical composition for treating a patient having cancercomprising a compound and a pharmaceutically acceptable enhancercarrier, diluent and/or excipient, wherein said compound is selectedfrom the group consisting of9-[2-(1-azabicyclo[2,2,2]3-oxooctyl)]-methyl-6-chloro-9H-purine,2,2-bis(hydroxymethyl-1-azabicyclo[2,2,2]octan-3-one, and2-hydroxymethyl-3,3-dihydroxy-1-azabicyclo[2,2,2]octane.
 2. Thecomposition according to claim 1, wherein said compound is2,2-bis(hydroxymethyl)-1-azabicyclo[2,2,2]octan-3-one.
 3. Thecomposition according to claim 1, wherein said compound is2-hydroxymethyl-3,3-dihydroxy-1-azabicyclo[2,2,2]octane.
 4. Thecomposition according to claim 1, wherein said compound is coupled to afunctional moiety.
 5. The composition according to claim 1, wherein saidcompound is9-[2-(1-azabicyclo[2,2,2]3-oxooctyl)]-methyl-6-chloro-9H-purine.